1. Field of the Invention
The present invention relates to a method for the isolation of nopaline and its analogues from diastereomers of these compounds.
2. Description of the Prior Art
Considerable activity exist in the area of genetic engineering of microorganisms. However, only recently has there been significant activity involving genetic engineering of higher plants. One method which has been proposed for introducing genetic material into higher plants involves use of the bacterium Agrobacterium tumefaciens to induce a crown gall tumor in a dicotyledoneous plant. Variant A. tumefaciens bacterial strains contain a large Ti (tumor-inciting) plasmid, part of which, a specific segment called the T-DNA (transferred DNA), integrates into the plant nuclear DNA where it is retained and expressed even after the tumors redifferentiate. Accordingly, the Ti plasmid is a possible vector for accomplishing genetic engineering in plants. See, Ream and Gordon, "Crown Gall Disease and Prospects for Genetic Manipulation of Plants", Science, 218: 854-859 (1982).
Crown gall tumor cells produce opines, which are unusual amino acid derivatives not found in normal plant cells. The ability of transformed cells to synthesize these amino acid derivatives depends strictly on the bacterial strain Which causes the tumor. Furthermore, bacteria which induce a specific amino acid derivative can utilize that derivative as a single source of carbon and nitrogen, but cannot utilize opines produced by tumors caused by other strains of bacteria. Accordingly, opines can be utilized in the preparation of bacterial growth media useful for the selection of appropriate strains of bacteria. Other derivatives are toxic to the bacteria containing the appropriate catabolic enzymes coded in the Ti plasmid. See Petit and Tempe, "Isolation of Agrobacterium Ti-plasmid Regulatory Mutants", Molec. Gen. Genet. 167:147-155 (1978). Use of these derivatives in selective media permits selection of mutant strains of bacteria which do not have the ability to catabolize the toxic derivatives.
Accordingly, a source of opines useful for producing the selective media is needed. While it is possible to isolate different opines from crown gall tumors of plants, the isolation techniques are tedious and a general synthetic method capable of synthesizing different opines in good yield is needed. See Fermin and Fenwick, Phytochemistry 16:761-762 (1977).
Synthetic procedures for the synthesis of opines and particularly for the synthesis of nopaline and its derivatives exist in the prior art but suffer from disadvantages caused by the difficulty of separating nopaline (or an analog of nopaline) from the diastereoisomer produced by the synthetic method, known as isonopaline (or the corresponding isonopaline analogue).
Cooper and Firmin, Org. Prep. Proced. Int., 9: 99-101 (1977) disclose a chemical synthesis of nopaline and isonopaline by the base catalyzed condensation of 2-oxoglutaric acid with L-arginine, followed by borohydride reduction of the resulting Schiff's base. The product was dissolved in boiling water and a precipitate was obtained uponstanding at 4.degree. C. However, because of the heating step, although unknown at the time, this product was not pure nopaline. The thus obtained precipitate was substantially contaminated at least with the cyclized derivative, or pyronopaline. In the same fashion, the precipitate obtained from the mother liquor by addition of ethanol, was not pure isonopaline, but contained at least some amount of the cyclized derivative, or pyroisonopaline.
Jensen et al, Biochemical and Biophysical Research Communications, 75: 1066-1070 (1977), disclose a similar reaction for synthesizing the nopaline diastereoisomers but using cyanoborohydride instead of borohydride which gave a 80% yield, substantially higher than that obtained with sodium borohydride. A weakly levorotatory nopaline was obtained in a 2-4% yield, which is presumed by the investigators to be a diostereoisomeric mixture of the weakly levorotatory natural nopaline and of the strongly dextrorotatory isonopaline having the L- configuration at both centers (see, reference, page 1069, last paragraph). However, as shown in the present application, both nopaline and isonopaline are strongly dextrorotatory when in substantially purified form. Thus, both the natural and synthetic nopaline of Jensen et al are substantially contaminated.
Hatanaka et al, Phytochemistry 21(1):225-227 (1981) disclose a variation of the cyanoborohydride reaction which gave a total yield of nopaline and isonopaline of 70%. In this method the separation of the diastereoisomers was accomplisned by chromatography on an anion exchange substrate.
However, the published analytical data of the results of all these separations indicate that purified nopaline substantially free of isonopaline was not obtained.
Firmin and Fenwick disclose a chemical synthesis of nopalinic acid by the aqueous hydrazinolysis of nopaline, followed by anion exchange chromatography. However, the product was obtained upon recrystallization from aqueous ethanol in a yield of only 42%.
Accordingly, an improved method for the separation of nopaline and its analogs from their diastereoisomers is necessary in order for these synthetic methods to be fully developed.